ER Pathway Research Articles

Abstract P6-20-08: Anti-tumor activity of elacestrant (RAD1901) in models harboring ESR1 mutations resistant to standard of care therapies

Abstract Background: Estrogen receptor positive (ER+) breast cancers make up approximately 75% of all breast cancers diagnosed and ER, a protein encoded by the ESR1 gene, plays a major role in the initiation, growth and survival of these cancers. Current targeted therapies inhibit the ER pathway by either blocking the synthesis of the natural ligand of ER, estradiol, (aromatase inhibitors (AI)), or by antagonizing and/or degrading the receptor (selective estrogen receptor modulators (SERMs) and selective estrogen receptor degraders (SERDs)). AIs are used in both the adjuvant and metastatic setting and recent clinical reports have shown that 20-50% of patients that had received AI therapy had detectable mutations in the ER ligand binding domain (ER-LBD). Two frequently found point mutations in the ER-LBD, Y537S and D538G, have been shown to result in estradiol-independence and constitutive activation of ER, consistent with their ability to cause resistance to AIs. While the selection of ESR1 mutations post-AI has been demonstrated clinically, the clinical response of ESR1 mutant tumors to fulvestrant, an approved SERD, is not fully understood. Preclinical studies have suggested that ESR1 mutations can cause decreased binding and a corresponding decrease in potency of ER antagonists, including fulvestrant (SERD) and tamoxifen (SERM). Conversely, clinical data from the SoFEA, PALOMA-3, and FERGI trials suggested the presence of ESR1 mutations did not alter fulvestrant activity. The limited clinical data that exists, however, is based on retrospective study designs with relatively small data sets, making it difficult to accurately predict fulvestrant activity against specific mutations and the activity of fulvestrant against tumors that harbor multiple mutations. In fact, recent additional data from the PALOMA-3 trial suggests that the Y537S mutation, specifically, was selected out in clinical samples from patients treated with fulvestrant, more closely matching preclinical results. This suggests there may be certain contexts of ESR1 mutations where fulvestrant may have limited activity. It will be important to further understand the consequence of specific mutations and to utilize therapies that have activity against all ESR1 mutations. We have previously described elacestrant (RAD1901), a novel orally bioavailable SERD, that exhibited activity in multiple ER+ breast cancer models. Interestingly, elacestrant exhibited similar effects to fulvestrant in in vitro ESR1 mutant models, however, in some in vivo PDX models harboring the Y537S mutation elacestrant inhibited growth, while fulvestrant had limited activity. Here, we describe a more complete in vivo dataset describing elacestrant activity versus fulvestrant in multiple patient-derived xenograft (PDX) models harboring ESR1 mutations. Methods: Multiple PDX models harboring natural mutations in ESR1 or genetically-engineered CRISPR models were used to assess the anti-tumor efficacy and the pharmacokinetic/pharmacodynamic properties of elacestrant and fulvestrant. Results: Elacestrant significantly inhibited the growth of xenograft models harboring ESR1 mutations, including those harboring Y537S or D538G mutations and models that were insensitive to fulvestrant and tamoxifen. Citation Format: Patel H, Tao N, Arlt H, Bihani T. Anti-tumor activity of elacestrant (RAD1901) in models harboring ESR1 mutations resistant to standard of care therapies [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-20-08.

Abstract OT1-01-02: An open-label, randomized, multi-center phase 2 study evaluating the activity of lasofoxifene relative to fulvestrant for the treatment of postmenopausal women with locally advanced or metastatic ER+/HER2 - breast cancer (MBC) with an ESR1 mutation

Abstract Endocrine based therapy is the standard treatment for estrogen receptor positive (ER+) MBC. Agents targeting the ER pathway including aromatase inhibitors (AIs), fulvestrant and tamoxifen along with CDK 4/6 inhibitors are considered standard for first and 2nd line treatment. However, endocrine resistance develops in nearly all patients and the optimal systemic therapy after progression on a CDK 4/6 inhibitor is unknown. Lasofoxifene is a third generation SERM previously investigated for the treatment of osteoporosis and vulvo-vaginal atrophy (VVA). In a large phase 3 trial evaluating the efficacy of lasofoxifene for the postmenopausal treatment of osteoporosis, lasofoxifene significantly reduced the incidence of ER+ breast cancer. Further unpublished preclinical data have demonstrated significant in vitro and in vivo efficacy in non-clinical breast cancer models including models with and without ESR1 mutants. Moreover, lasofoxifene significantly reduced metastases in ESR1 mutated models. These non-clinical and clinical data provide a strong rationale to pursue a phase 2 clinical trial in women with ER+, ESR1 mutated MBC. This open-label, multi-center study will compare the efficacy and tolerability of lasofoxifene (5 mg orally daily) to fulvestrant (IM 500 mg D1,15,29 and then q30 D) in a 1:1 randomization. Inclusion criteria include postmenopausal women with ER+ advanced breast cancer; progression on a non-steroidal AI in combination with a CDK 4/6 inhibitor; and a known ESR1 mutation. Approximately 90 patients with measurable or evaluable disease (i.e. bone only) will be recruited to have at least 40 patients per treatment arm. The primary endpoint will be progression free survival (PFS) with secondary endpoints of objective response rate (ORR), clinical benefit rate (CBR), duration of response (DoR) and time to response (TTR). It is assumed that lasofoxifene will double the median PFS compared to fulvestrant in this ESR1 mutation patient population for a hazard ratio 0.5 and a power of 89% to reach a 1-sided p of <0.05. The study will commence in 4Q2018 and will complete recruitment in 1 year. It is anticipated that 25-30 centers in the US will be participating. Citation Format: Plourde PV, Schwartzberg LS, Greene GL, Portman DJ, Komm BS, Jenkins SN, Liu P-Y, Portman MD, Goetz MP. An open-label, randomized, multi-center phase 2 study evaluating the activity of lasofoxifene relative to fulvestrant for the treatment of postmenopausal women with locally advanced or metastatic ER+/HER2 - breast cancer (MBC) with an ESR1 mutation [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr OT1-01-02.

Extended Adjuvant Therapy with Neratinib Plus Fulvestrant Blocks ER/HER2 Crosstalk and Maintains Complete Responses of ER+/HER2+ Breast Cancers: Implications to the ExteNET Trial.

The phase III ExteNET trial showed improved invasive disease-free survival in patients with HER2+ breast cancer treated with neratinib versus placebo after trastuzumab-based adjuvant therapy. The benefit from neratinib appeared to be greater in patients with ER+/HER2+ tumors. We thus sought to discover mechanisms that may explain the benefit from extended adjuvant therapy with neratinib.Experimental Design: Mice with established ER+/HER2+ MDA-MB-361 tumors were treated with paclitaxel plus trastuzumab ± pertuzumab for 4 weeks, and then randomized to fulvestrant ± neratinib treatment. The benefit from neratinib was evaluated by performing gene expression analysis for 196 ER targets, ER transcriptional reporter assays, and cell-cycle analyses. Mice receiving "extended adjuvant" therapy with fulvestrant/neratinib maintained a complete response, whereas those treated with fulvestrant relapsed rapidly. In three ER+/HER2+ cell lines (MDA-MB-361, BT-474, UACC-893) but not in ER+/HER2- MCF7 cells, treatment with neratinib induced ER reporter transcriptional activity, whereas treatment with fulvestrant resulted in increased HER2 and EGFR phosphorylation, suggesting compensatory reciprocal crosstalk between the ER and ERBB RTK pathways. ER transcriptional reporter assays, gene expression, and immunoblot analyses showed that treatment with neratinib/fulvestrant, but not fulvestrant, potently inhibited growth and downregulated ER reporter activity, P-AKT, P-ERK, and cyclin D1 levels. Finally, similar to neratinib, genetic and pharmacologic inactivation of cyclin D1 enhanced fulvestrant action against ER+/HER2+ breast cancer cells. These data suggest that ER blockade leads to reactivation of ERBB RTKs and thus extended ERBB blockade is necessary to achieve durable clinical outcomes in patients with ER+/HER2+ breast cancer.

Adenosine Attenuates LPS-Induced Cardiac Dysfunction by Inhibition of Mitochondrial Function via the ER Pathway.

Sepsis is a life-threatening organ dysfunction syndrome with a high rate of mortality. It is caused by an abnormal immune response to infection, and the occurrence of sepsis-induced cardiomyopathy is the primary cause of death. The present study was designed to examine the effects of adenosine on lipopolysaccharide- (LPS-) induced cardiac anomalies and the underlying mechanisms involved. Adenosine (25, 50, and 100 mg/kg, i.g., 2 times/day) was administered for three days, followed by the induction of sepsis by intraperitoneal injection of LPS (10 mg/kg/2h). The effects of adenosine on inflammatory factors, LVEF, LVFS, and MAPK in septic rats (half male and half female) were observed. Subsequently, the effect of adenosine (10 μM) on the mitochondrial function of H9c2 cells stimulated with LPS (20 μg/mL, 24 h) was observed in the presence and absence of the estrogen receptor-specific antagonist ICI182,780. The results show that medium to high doses of adenosine can significantly promote cardiac function (LVEF and LVFS) and reduce the levels of inflammatory factors (TNF-α, IL-6, PCT, and cTnI) and p-JNK in septic rats, with a significant difference seen between male and female rats. The results of flow cytometry show that adenosine significantly inhibited increases in ROS levels, mitochondrial membrane potential, and the swelling degree of mitochondria in H9c2 cells stimulated with LPS, but this effect could be blocked by ICI182,780, indicating that adenosine attenuated LPS-induced cardiac dysfunction by inhibiting mitochondrial function via the ER pathway.

Combined Blockade of Activating ERBB2 Mutations and ER Results in Synthetic Lethality of ER+/HER2 Mutant Breast Cancer.

We examined the role of ERBB2-activating mutations in endocrine therapy resistance in estrogen receptor positive (ER+) breast cancer. ERBB2 mutation frequency was determined from large genomic databases. Isogenic knock-in ERBB2 mutations in ER+ MCF7 cells and xenografts were used to investigate estrogen-independent growth. Structural analysis was used to determine the molecular interaction of HER L755S with HER3. Small molecules and siRNAs were used to inhibit PI3Kα, TORC1, and HER3. Genomic data revealed a higher rate of ERBB2 mutations in metastatic versus primary ER+ tumors. MCF7 cells with isogenically incorporated ERBB2 kinase domain mutations exhibited resistance to estrogen deprivation and to fulvestrant both in vitro and in vivo, despite maintaining inhibition of ERα transcriptional activity. Addition of the irreversible HER2 tyrosine kinase inhibitor neratinib restored sensitivity to fulvestrant. HER2-mutant MCF7 cells expressed higher levels of p-HER3, p-AKT, and p-S6 than cells with wild-type HER2. Structural analysis of the HER2 L755S variant implicated a more flexible active state, potentially allowing for enhanced dimerization with HER3. Treatment with a PI3Kα inhibitor, a TORC1 inhibitor or HER3 siRNA, but not a MEK inhibitor, restored sensitivity to fulvestrant and to estrogen deprivation. Inhibition of mutant HER2 or TORC1, when combined with fulvestrant, equipotently inhibited growth of MCF7/ERBB2 V777L xenografts, suggesting a role for TORC1 in antiestrogen resistance induced by ERBB2 mutations. ERBB2 mutations hyperactivate the HER3/PI3K/AKT/mTOR axis, leading to antiestrogen resistance in ER+ breast cancer. Dual blockade of the HER2 and ER pathways is required for the treatment of ER+/HER2 mutant breast cancers.

Relation of EGFR/PI3K/AKT signaling components with tamoxifen efficacy in patients with estrogen-dependent breast cancer

Background. It is generally accepted that crosstalk between the growth factor receptor and ER pathways implicated in tamoxifen resistance. The aim of the study was to examine the relationship between mRNA level, protein expression and gene polymorphism of the EGFR/PI3K/AKT signaling components with tamoxifen efficacy in patients with estrogen-dependent breast cancer. Materials and methods. The study included 95 breast cancer patients who had received adjuvant tamoxifen, of which 31 patients developed recurrence/metastasis after tamoxifen treatment (tamoxifen resistance group), 64 patients did not develop any diseases progression (tamoxifen sensitive group) during the 5 years of follow-up. Genotypes for ESR1 (rs2077647, rs2228480, rs1801132), EGFR (rs1468727, rs2227983), AKT1 (rs1130233) and PTEN (rs11202592) were analyzed using a TaqMan assay. Using reverse transcription-PCR, the relative expression of mRNA for ESR1, EGFR, AKT1 and PTEN was determined. ERα, EGFR, Akt (pS473) and PTEN expression level was evaluated using immunohistochemistry. Progression-free survival (PFS) was estimated by Kaplan – Meier analysis. Results. The minor allele of ESR1 rs2077647 was more prevalent in tamoxifen sensitive tumors compared to tamoxifen resistant tumors (p = 0.044). We found high AKT1 mRNA expression level in tamoxifen sensitive group compared with tamoxifen resistance patients (7.27 ± 5.29 and 0.02 ± 0.01, respectively, p = 0.014). ESR1 rs2228480 was significantly associated with tamoxifen resistance (p = 0.028). EGFR and Akt (pS473) protein expression level was significantly higher in the tamoxifen resistance group compared to tamoxifen sensitive breast cancer patients (p = 0.006 and 0.037, respectively). Patients carrying mutant genotypes of ESR1 rs2228480 had a poorer progression-free survival than those carrying wild and heterozygous variants (log rank p = 0.043). Positive EGFR tumor expression as well as positive Akt (pS473) expression were significantly associated with shorter PFS (log rank p = 0.014 and 0.048, respectively). Conclusion. Polymorphic sites of the ESR1 gene, AKT1 mRNA expression, EGFR expression level and Akt (pS473) protein expression can be potential molecular markers associated with tumor sensitivity/resistance to tamoxifen treatment.

Meta-microRNA Biomarker Signatures to Classify Breast Cancer Subtypes.

Breast cancer is one of the leading causes of morbidity and mortality that is in need of novel diagnostics and therapeutics. Meta-analysis of microarray data offers promise to combine studies and provide more robust results. We report here a molecular classification of pathological subtypes (estrogen receptor [ER], progesterone receptor [PR], and Human Epidermal Growth Factor Receptor 2 [HER2]) of breast cancers with microRNA (miRNA)-dependent signatures. A ranking-based meta-analysis approach was applied to eight independent microarray data sets and meta-miRNA lists were obtained that are specific to each breast cancer subtype. The comparison of the lists with miRCancer and the PhenomiR 2.0 databases pointed out nine prominent miRNAs: let-7b-5p, let-7c-5p, let-7e-5p, miR-130a-3p, miR-30a-5p, miR-92a-1-5p, miR-211-5p, miR-500a-3p, and miR-516b-3p. Further analysis conducted with the TCGA data showed that these miRNAs can differentiate tumors from normal samples as well as discriminate the molecular subtypes of breast cancer. According to the PAM50 classification, three of these miRNAs (let-7b-5p, let-7c-5p, and miR-30a-5p) downregulated significantly, whereas miR-130a-3p, miR-92a-1-5p, miR-211-5p, and miR-500a-3p upregulated in tumors from the luminal A to the basal-like subtypes. When the prominent meta-miRNAs and their targets were analyzed, they appeared to be taking part in important signaling pathways in cancer such as the PI3K-Akt signaling and the p53 signaling pathways. Furthermore, the regulatory genes, which are key players for ER, PR, and ErBb signaling pathways, were found to be under control of several meta-miRNAs. These meta-miRNAs and the genes they are regulating offer new promise for future translational research and potential targets for precision medicine diagnostics.

Abstract IA26: Clinical development of PI3K/AKT pathway inhibitors in breast cancer

Abstract Targeting the PI3K/AKT/mTOR pathway has been a focus for drug development since the discovery of PI3K-activating mutations in p110α over 10 years ago. Breast cancer has been a primary indication for development of this class of drugs because of the high prevalence of these mutations, up to 45% in hormone receptor-positive breast cancer, and the synergy observed in combinations with endocrine therapies. An mTOR inhibitor, everolimus, was approved in breast cancer in combination with exemestane in 2012, indicating the synergy between ER and PI3K pathway translates to the clinic. Apitolisib, a dual PI3K pan-isoform and mTOR inhibitor, has discontinued development due to toxicities, despite compelling preclinical activity across multiple tumor types regardless of PI3K and PTEN status. Pictilisib, a pan-isoform PI3K inhibitor, has shown some evidence of activity, but in phase II studies has demonstrated that the activity is limited by dose intensity. Buparlisib, a pan-isoform PI3K inhibitor, has demonstrated efficacy in a large phase III study, but was not deemed sufficiently active relative to the toxicity in HR+ breast cancer to be submitted for regulatory approval. Second-generation PI3K and AKT inhibitors with higher selectivity and mutant specificity are in clinical development and hope to thread a better therapeutic index by dosing higher for more profound inhibition, and/or selectively inhibiting mutated forms of PI3K. Taselisib (GDC-0032) is a beta-sparing and PI3K-mutant selective inhibitor that leads to degradation of only the mutant form of PI3K and is currently in phase III. More recently, taselisib met the primary endpoint of improved response rates in a randomized, neoadjuvant phase II trial, LORELEI. Alpelisib is an alpha-selective PI3K inhibitor that is also in phase III. Recent data with ipatasertib, an AKT inhibitor, has demonstrated better tolerability and efficacy in randomized phase II trials of both prostate cancer and triple-negative breast cancer, where the PI3K/AKT signaling pathway is activated by either loss of PTEN or PI3K mutations, and may offer a better therapeutic index given its unique mechanism of action and place in the PI3K signaling pathway. Overall, the PI3K/AKT pathway is still considered an important pathway to target for certain cancers; however, target selectivity, dosing, and the right combination are critical to find a therapeutic index to provide a good option for patients. Citation Format: Mika Derynck. Clinical development of PI3K/AKT pathway inhibitors in breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr IA26.

Neratinib: Inching Up on the Cure Rate of HER2+ Breast Cancer?

Neratinib was recently approved by the FDA for extended adjuvant treatment of HER2+ breast cancer. The ExteNET trial showed improvement in invasive disease-free survival (iDFS) in the neratinib arm compared with placebo. The benefit was more pronounced in patients with estrogen receptor-positive (ER+)/HER2+ tumors, suggesting bidirectional cross-talk between the ER and HER pathways. Clin Cancer Res; 24(15); 3483-5. ©2018 AACRSee related article by Singh et al., p. 3486.

Genetic variation in Wnt/β-catenin and ER signalling pathways in female and male elite dancers and its associations with low bone mineral density: a cross-section and longitudinal study.

Some athletes (e.g. gymnasts, dancers, swimmers) are at increased risk for low bone mineral density (BMD) which, if untreated, can lead to osteoporosis. To investigate the association of genetic polymorphisms in the oestrogen receptor (ER) and the Wnt/β-catenin signalling pathways with low BMD in elite and pre-elite dancers (impact sport athletes). The study included three phases: (1) 151 elite and pre-elite dancers were screened for the presence of low BMD and traditional osteoporosis risk factors (low body weight, menstrual disturbances, low energy availability); (2) a genetic association study was conducted in 151 elite and pre-elite dancers and age- and sex- controls; (3) serum sclerostin was measured in 101 pre-elite dancers and age- and sex-matched controls within a 3-year period. Eighty dancers revealed low BMD: 56.3% had at least one traditional osteoporosis risk factor, whereas 28.6% did not display any risk factor (37.2% revealed traditional osteoporosis risk factors, but had normal BMD). Body weight, menstrual disturbances and energy availability did not fully predict bone mass acquisition. Instead, genetic polymorphisms in the ER and Wnt/β-catenin pathways were found to be risk factors for low BMD in elite dancers. Sclerostin was significantly increased in dancers compared to controls during the 3-year follow-up (p < 0.05). Elite and pre-elite dancers demonstrate high prevalence of low BMD, which is likely related to genetic variants at the Wnt/β-catenin and ER pathways and not to factors usually associated with BMD in athletes (body weight, menstrual disturbances, energy deficiency).

Abstract 3690: Measuring functional signal transduction pathway activity on breast cancer tissue samples to determine intra-tumor heterogeneity and heterogeneity between primary and metastatic tumors

Abstract Background Treatment with targeted drugs is increasingly used in breast cancer aiming to block the tumor driving pathway(s). Drug choice is often based on a single primary tumor biopsy. It is important to ascertain that biopsied tumor material is representative for the tumor or metastases to treat with respect to tumor driving signaling pathway. Little is known about pathway heterogeneity within primary breast cancer, and between primary and metastatic tumors. A novel analysis method was developed to identify and quantify activity of signal transduction pathways in cancer tissue, based on Bayesian models which infer a pathway activity score from transcription factor target gene mRNA levels (Cancer Res 2014 Jun 1;74(11):2936-45). Methods Originally for AffymetrixU133Plus2.0, the pathway analysis was adapted for RT-qPCR enabling use on FFPE tissue. Calibration was performed using tissue samples with known pathway activity. Functional pathway activity of ER, AR, PI3K-FOXO, Hedgehog (HH), TGFbeta, and Wnt pathways was analyzed to assess (1) intra-tumor heterogeneity: 2-5 FFPE blocks of 17 primary breast cancers (9 luminal A, 4 luminal B, 1 HER2, 3 triple negative (TN), as defined by surrogate immunohistochemistry); (2) heterogeneity between primary and associated metastases: 9 patients with FFPE blocks from primary and 12 metastases. Results Intra-tumor heterogeneity: In 11 out of 13 Luminal A- and B-like tumors the ER pathway was active in all samples, 1 showed ER pathway heterogeneity; 11 had an active TGFbeta pathway, heterogeneic in 3; in one ER inactive tumor one (out of 5) samples was AR active. In 2 TN tumors TGFbeta and PI3K pathways were combined active, in one TN tumor heterogeneic TGFbeta activity was observed. PI3K pathway activity increased with malignancy grade and showed most intra-tumor variation. Overall, pathway heterogeneity was detected in over one third of tumors, least in Luminal A-like (2/9) compared to Luminal B-like (3/4) and triple negative (1/3) tumors. Extensive heterogeneity was found between primary breast cancer and metastases, and between metastatic locations of the same patient. ER pathway activity decreased in 5/9 patients and increased in 4/9 patients in one or more metastatic tumors; PI3K became active in metastasis of 2 patients and inactive in 1; the Wnt pathway became active in 3 patients in bone and ileum metastases; TGFbeta was lost in 5 patients, and became active in 1 bone metastasis; HH became active in 1 ovarian and AR in 1 bone metastasis. Conclusion Intra-tumor heterogeneity was lower in ER active compared to TN breast cancer, suggesting a need for multiple biopsies to adequately characterize TN for neoadjuvant therapy. Between primary tumor and metastases, heterogeneity was extensive indicating the need for pathway analysis on metastatic tumors prior to targeted treatment. Citation Format: Anja Van De Stolpe, Anne van Brussel, Cathy Moelans, Marcia A. Inda, Wim Verhaegh, Eveline den Biezen, Paul van Diest. Measuring functional signal transduction pathway activity on breast cancer tissue samples to determine intra-tumor heterogeneity and heterogeneity between primary and metastatic tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3690.

Abstract 1052: Identification of signal transduction pathway activity in patient-derived xenograft models in comparison with their originating clinical samples of a variety of human cancer types

Abstract Background. Targeted drugs are directed against cellular signal transduction pathways, such as the PI3K pathway, and efficacy depends on the active pathway in the tumor. Development and clinical application of targeted drugs for personalized cancer treatment require tests which identify pathway activity in cancer cells/tissue. Patient-derived xenograft (PDX) models are becoming the cornerstone of preclinical drug development. Characterization of PDX models and clinical samples for pathway activity is needed for successful preclinical drug testing and patient drug response prediction. Inference of signal transduction pathway activity from cancer genomics information is often unreliable. A novel analysis method has been developed which enables identification and quantification of activity of signal transduction pathways, based on Bayesian reasoning models which infer an activity score from mRNA levels of pathway target genes (Verhaegh et al, Cancer Res 2014:2936-45). For the AR, ER, PI3K, Hedgehog, TGFbeta, Wnt, and NFkB pathways, models have been biologically validated on a number of different cell/tissue types. Methods. For a number of different cancer types (among which colon, breast, lung, ovarian, bladder, osteosarcoma, melanoma, esophagus), AR, ER, PI3K, Hedgehog, TGFbeta, Wnt, and NFkB pathway activity was inferred from Affymetrix HG-U133Plus2.0 microarray data, available from clinical studies (GEO database) and from PDX models (Charles River). Clinical and xenograft pathway activity scores were compared and where possible correlated to genomic datato identify associated pathway driving alterations. Results. Different cancer types were analyzed and compared. Pathway activity patterns for different cancer types in PDX models resemble pathway activity in clinical patient cohorts. Furthermore, known associated cancer mutations are reproduced in PDX models, such as APC mutations in colon PDX and ovary PDX, and an activating beta-catenin mutation in breast PDX, that show an activated Wnt pathway. Conclusion. Signal transduction pathway activity in PDX models resembles pathway activity in patient cohorts with corresponding cancer types and corresponds with known driving mutations. Functional pathway activity testing in PDX models is expected to increase xenograft model utility for (1) further validation of pathway tests for clinical diagnostic use and for (2) preclinical testing of targeted drug efficacy. Citation Format: Wim Verhaegh, Anja van de Stolpe, Nevisa Caushaj, Manuel Landesfeind, Angelika Zaremba. Identification of signal transduction pathway activity in patient-derived xenograft models in comparison with their originating clinical samples of a variety of human cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1052.

Abstract 2173: Testing neratinib-containing drug combination regimens on HER2 mutated non-amplified, ER+ breast cancer patient-derived xenografts

Abstract Introduction: HER2 activating mutations are a novel, druggable genomic alteration in metastatic breast cancer (MBC). These HER2 mutations are predominantly found in HER2 gene amplification negative, hormone receptor positive breast cancers. We have previously demonstrated that HER2 mutations can be potently inhibited by the second generation, irreversible pan-HER tyrosine kinase inhibitor, neratinib (Bose et al., Cancer Discovery 2013). Further, we performed a phase II clinical trial to treat HER2 mutated MBC and we found that neratinib monotherapy produced a clinical benefit rate of 31% and progression free survival of 16 weeks in a heavily pre-treated patient population (Ma et al., Clinical Cancer Research 2017). The objective of the current pre-clinical study is to develop combination strategies to improve the anti-tumor activity of neratinib in HER2 mutated breast cancer. As ER and CDK4/6 signaling activation are known resistance mechanisms to HER2 targeted agents in HER2 positive breast cancer, we hypothesized that neratinib in combination with agents that target ER (fulvestrant) or CDK4/6 (palbociclib) will induce synergistic anti-tumor effect in HER2 mutated MBC. In addition, we hypothesized that dual HER2 targeting (with trastuzumab) or in combination with chemotherapy agents (vinorelbine), may be more effective than neratinib alone. Since these partnering agents are well established in the treatment of breast cancer and are without overlapping toxicities with neratinib, rapid clinical translation is possible for the treatment of patients with HER2 mutated, ER+ MBC. Description of our Human-in-Mouse Patient Derived Xenografts (PDX): We developed two HER2 mutated breast cancer PDX lines. Both lines come from HER2 mutation positive, HER2 gene amplification negative, ER+ MBC patients. PDX-51 has a HER2 exon 20 insertion mutation (ERBB2 G776insYVMA), a PIK3CA H1047R mutation and is wild-type for TP53. PDX-64 has a HER2 kinase domain missense mutation (ERBB2 L869R) and is wild-type for both PIK3CA and TP53. Both PDX lines are grown in female NSG strain mice without any exogenous estrogen supplementation. Results: We tested combinations of neratinib with fulvestrant, palbociclib, trastuzumab, or vinorelbine for their anti-tumor activity and effects on cell proliferation and survival pathway signaling activities, including ER, CDK4/6 and HER2 pathways, in vivo. We will present data on the efficacy of these drug combinations. Tumor size measurements and reverse phase protein array (RPPA) data on the treated PDX lines will be shown. Citation Format: Shunqiang Li, Tina M. Primeau, Stephanie L. Pratt, Katherine R. Harrill, Francesca Avogadri-Connors, Alshad S. Lalani, Cynthia X. Ma, Ron Bose. Testing neratinib-containing drug combination regimens on HER2 mutated non-amplified, ER+ breast cancer patient-derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2173.

Abstract 943: SAR439859, an orally bioavailable selective estrogen receptor degrader (SERD) that demonstrates robust antitumor efficacy and limited cross-resistance in ER+ breast cancer

Abstract Despite resistance to endocrine therapies, estrogen receptor-positive (ER+) breast cancers (BC) still rely on ER. Eliminating ER by inducing its degradation with selective ER downregulators (SERD) should induce complete ablation of ER pathways. The clinical SERD fulvestrant, although it has demonstrated clinical benefits, is hampered by its poor drug properties and undesirable pharmacokinetics, undermining its optimal clinical activity. Therefore there is an unmet need for an improved therapy targeting ER. Here we describe SAR439859, a novel, nonsteroidal, orally bioavailable SERD currently in clinical trials. SAR439859 has a potent ER antagonist and ER degrading activity that translates in a robust inhibition of ER signaling in multiple ER+ breast cancer cell lines, including tamoxifen-resistant lines as well as cell lines harboring ER mutations. SAR439859 displays a broad and superior ER degradation activity across a large panel of ER+ cells. Importantly, based on its mechanism of action SAR439859 shows limited cross-resistance with other clinical SERD molecules. SAR439859 induces strong in vivo antitumor activity against a variety of BC cell lines and patient-derived xenografts, including models that harbor ERα mutations. The transcriptional profile analysis highlighted a crosstalk of ER mutant signaling and other oncogenic pathways. Interestingly, CDK4/6 inhibition by palbociclib induces partial activation of ER pathways as potential mechanism of tumor escape, which is completely abolished by the combination of SAR439859 with palbociclib. Finally, we demonstrate that SAR439859 in combination with palbociclib can lead to higher in vivo efficacy. This study highlights novel mechanism of ER degradation by SAR439859 that leads to profound inhibition of ER signaling as well as modulation of other oncogenic pathways and provides rationale for the ongoing clinical investigation of SAR439859 in ER+ breast cancer patients, both as a single agent and in combination with approved agents, such as CDK4 inhibitor. Citation Format: Monsif Bouaboula, Maysoun Shomali, Jane Cheng, Natalia Malkova, Fangxian Sun, Malvika Koundinya, Zhuyan Guo, Stephane Poirier, Mikhail Levit, Dietmar Hoffman, Hui Cao, Laurent Bestret, Francisco Adrian, Christoph Winter, Youssef El-Ahmad, Sylvie Vincent, Frank Halley, Gary McCort, Laurent Schio, Vicky Richon, Hong Cheng, Karl Hsu, Chris Soria, Patrick Cohen, Joanne Lager, Carlos Garcia-Echeverria, Laurent Debussche. SAR439859, an orally bioavailable selective estrogen receptor degrader (SERD) that demonstrates robust antitumor efficacy and limited cross-resistance in ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 943.

New insights into the mechanism of phthalate-induced developmental effects

To investigate the biological pathways involved in phthalate-induced developmental effects, zebrafish embryos were exposed to different concentrations of di-(2-ethylhexyl) (DEHP) and di-butyl phthalate (DBP) for 96 h. Embryonic exposure to DEHP and DBP induced body length decrease, yolk sac abnormities, and immune responses (up-regulation of immune proteins and genes). The lipidomic results showed that at a concentration of 50 μg/L, DEHP and DBP significantly reduced the levels of fatty acids, triglycerides, diacylglycerol, and cholesterol. These effects are partly explained by biological pathway enrichment based on data from the transcriptional and proteomic profiles. Co-exposure to DBP and ER antagonist did not significantly relieve the toxic symptoms compared with exposure to DBP alone. This indicates that phthalate-induced developmental abnormities in zebrafish might not be mediated by the ER pathway. In conclusion, we identified the possible biological pathways that mediate phthalate-induced developmental effects and found that these effects may not be driven by estrogenic activation.

Targeting tumour re-wiring by triple blockade of mTORC1, epidermal growth factor, and oestrogen receptor signalling pathways in endocrine-resistant breast cancer

BackgroundEndocrine therapies are the mainstay of treatment for oestrogen receptor (ER)-positive (ER+) breast cancer (BC). However, resistance remains problematic largely due to enhanced cross-talk between ER and growth factor pathways, circumventing the need for steroid hormones. Previously, we reported the anti-proliferative effect of everolimus (RAD001-mTORC1 inhibitor) with endocrine therapy in resistance models; however, potential routes of escape from treatment via ERBB2/3 signalling were observed. We hypothesised that combined targeting of three cellular nodes (ER, ERBB, and mTORC1) may provide enhanced long-term clinical utility.MethodsA panel of ER+ BC cell lines adapted to long-term oestrogen deprivation (LTED) and expressing ESR1wt or ESR1Y537S, modelling acquired resistance to an aromatase-inhibitor (AI), were treated in vitro with a combination of RAD001 and neratinib (pan-ERBB inhibitor) in the presence or absence of oestradiol (E2), tamoxifen (4-OHT), or fulvestrant (ICI182780). End points included proliferation, cell signalling, cell cycle, and effect on ER-mediated transactivation. An in-vivo model of AI resistance was treated with monotherapies and combinations to assess the efficacy in delaying tumour progression. RNA-seq analysis was performed to identify changes in global gene expression as a result of the indicated therapies.ResultsHere, we show RAD001 and neratinib (pan-ERBB inhibitor) caused a concentration-dependent decrease in proliferation, irrespective of the ESR1 mutation status. The combination of either agent with endocrine therapy further reduced proliferation but the maximum effect was observed with a triple combination of RAD001, neratinib, and endocrine therapy. In the absence of oestrogen, RAD001 caused a reduction in ER-mediated transcription in the majority of the cell lines, which associated with a decrease in recruitment of ER to an oestrogen-response element on the TFF1 promoter. Contrastingly, neratinib increased both ER-mediated transactivation and ER recruitment, an effect reduced by the addition of RAD001. In-vivo analysis of an LTED model showed the triple combination of RAD001, neratinib, and fulvestrant was most effective at reducing tumour volume. Gene set enrichment analysis revealed that the addition of neratinib negated the epidermal growth factor (EGF)/EGF receptor feedback loops associated with RAD001.ConclusionsOur data support the combination of therapies targeting ERBB2/3 and mTORC1 signalling, together with fulvestrant, in patients who relapse on endocrine therapy and retain a functional ER.

Androgen receptor expression in circulating tumor cells of metastatic breast cancer patients.

1087Background: The androgen receptor (AR) is of clinical relevance in metastatic breast cancer (mBC): AR has been associated with resistance to endocrine therapy and could be a potential target for therapy, especially in the triple negative (TN) subtype. A minimal-invasive way to determine AR expression is by characterization of circulating tumor cells (CTCs). We therefore assessed AR mRNA expression in CTCs (CTC-AR) from mBC patients representing different breast cancer subtypes in relation to outcome on endocrine therapy and 25 genes related to the ER and AR pathways. Furthermore, we assessed AR in matched primary tumors and CTC samples taken at advanced disease. Methods: AR and AR- and ER-related gene expression levels were measured in CellSearch-enriched CTCs from 133 mBC patients with ≥5 CTCs and in 48 matched formalin-fixed paraffin embedded primary tissues using quantitative reverse-transcriptase PCR. AR was considered positive if the expression was 1 standard deviation higher than the expression ...

Hormetic Response of H1299 Proliferation to Extracts of Hydnora Johannis Becc (Kausen Kasa) is Mediated Via Estrogen Receptor/EGFR and PKC

Hydnora Johannis Becc (HJ) is a herbal plant used in treating menopausal symptoms and other diseases in different countries, but the mechanism of actions are not known. The effect of HJ (0.1–2 mg/ml) extract on the proliferation of H1299 cell line was investigated. Cells were plated in 96 well plates and the effects of HJ were evaluated using MTT assay. Treatment of H1299 with HJ for 24 hours induced a dose‐dependent increase in cell proliferation increasing growth by 165%, 188%, 203% and 263% at 0.1, 0.25, 0.5 and 2 mg/ml, respectively compared to the control. Paradoxically at 48 hrs, the proliferative effects of the extract were turned into antiproliferative reducing cell growth by −74%, −65%, −37%, −17%, and 108% for 0.1, 0.25, 0.5, 1 and 2 mg/ml, respectively compared to the control. This effects showed a time‐dependent hermetic responses of H1299 to the HJ treatment. To investigate the mechanism(s) by which HJ regulate H1299 growth, cells were treated with HJ in the presence or absence of inhibitors of estrogen receptor (ER; Tamoxifen 10 and 20μg/mL), epidermal growth factor receptor (EGFR; Tyrphostin 5 μM) or Protein Kinase C (PKC; Calphostin 5 μM) and the effects on cell proliferation determined. Inhibition of ER, EGFR, and PKC significantly attenuated the proliferative effects of HJ treatment at 24 hrs. The antiproliferative effects observed at 48 hrs following HJ treatment were reverted to proliferation in the presence of inhibitors. These results suggest that HJ displays paradoxical phenomena which may be mediated by activation/inhibition of both proliferative and apoptotic signaling pathways in H1299 and mediated through interaction with ER, EGFR, and PKC signaling pathways. Further studies are warranted to investigate other signaling pathways involved in the Hydnora Johannis Becc actions.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of glutathione‐S‐transferases in the metabolism of the anti‐cancer agent and aromatase inhibitor, exemestane

Breast cancer is the most commonly diagnosed cancer in women, with approximately 1 in 8 women in the U.S. developing breast cancer over their lifetime. The majority of breast cancers diagnosed each year are estrogen receptor positive (ER+). Drugs targeting the ER pathway are a major treatment modality for ER+ patients and are additionally prescribed for the prevention of breast cancer. Exemestane (EXE) is an inhibitor which blocks the enzyme aromatase in the final step of estrogen biosynthesis. Studies examining the long‐term use of EXE as a chemopreventative agent demonstrated a reduction in breast cancer incidence by more than 65%. However, response and toxicity varied widely among individuals, which may be attributed to genetic polymorphisms in enzymes that metabolize EXE. Data from our lab demonstrate that a cysteine conjugates of EXE and its active metabolite 17β‐dihydro‐EXE (17β‐DHE) make up 77% of the total metabolite in urine. Cysteine conjugates are formed from metabolism of glutathione (GSH) conjugates in a reaction catalyzed by the glutathione‐S‐transferases (GST) family of the enzymes. The goal of the present study was to identify hepatic GSTs active in the metabolism of EXE and 17β‐DHE and to determine kinetic parameters for the identified enzymes. In a screening of the Protein Atlas, a total of 12 cytosolic and 3 microsomal GSTs were found to be hepatically expressed. EXE‐GSH conjugation activity in human liver samples was screened by incubating GSH with EXE or 17β‐DHE (0.125–1.0 mM) in cytosol (HLC) or microsomes (HLM) prepared from human livers. GSH conjugates were formed for both cellular fractions, indicating that both cytosolic and microsomal GST enzymes are active against EXE and 17β‐DHE. GSH conjugates were detected by UPLC‐MS and quantified using synthesized EXE‐GS and 17β‐DHE‐GS standards. The purity and identity of both standards and internal standards were confirmed using NMR and UPLC‐MS and found to be &gt; 95% pure. Individual recombinant human GSTs were then screened to determine which enzyme is primarily responsible for the metabolism of EXE. Commercially available cytosolic GSTs were purchased, and their activity was verified with a common GST substrate, 1‐chloro‐2.4‐dinitrobenzene. Results from the individual EXE‐GSH conjugation assays indicate the cytosolic enzymes GSTA1, GSTM3 and GSTP1 are active against EXE and 17β‐DHE; studies of microsomal GSTs are on‐going. These data suggest that several GSTs, both cytosolic and microsomal, may be important in the phase II conjugation of EXE and its major active metabolite, 17β‐DHE.Support or Funding InformationThis study is supported by National Institutes of Health (NIH) (Grant R01‐ES025460 and R01‐CA164366) and the Health Sciences and Services Authority of SpokaneThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Oestrogen-related receptor reduces vitellogenin expression by crosstalk with the ecdysone receptor pathway in female silkworm, Bombyx mori.

Oestrogen-related receptor (ERR) is involved in oestrogen receptor (ER) signalling pathways owing to its similarity to ER in terms of domain structure and co-activator and response elements. Although insects lack ER, they harbour an ERR gene that is thought to modulate metabolism and energy conversion via an unknown mechanism. The present study investigated the function of ERR in insects using female silkworm (Bombyx mori, Bm). We found that the expression of B.mori vitellogenin (BmVg) and B.mori ERR (BmERR) in the fat bodies of female silkworms at different stages of development exhibited alternating patterns, and RNA interference of BmERR in females induced BmVg transcription, resulting in an increase in egg weight relative to the control. Furthermore, BmERR was found to be involved in regulating the transcription of BmVg through an oestrogen-related receptor response element (ERRE) in the promoter of the BmVg gene, as demonstrated by electrophoretic mobility shift assay, cell transfection assay and chromatin immunoprecipitation. In summary, our results indicate that BmERR bound to the ERRE motif in the BmVg promoter reducing the expression of BmVg in the fat body of the female silkworm. To our surprise, the ERRE also showed the ability to bind the ecdysone receptor (BmEcR) and ultraspiracle complex. Thus, we surmise that ERR participates in steroid hormone signalling by engaging in crosstalk with the ER pathway in vertebrates and with the EcR pathway in insects.